Project Summary/Abstract Streptococcus mutans is the major etiological agent of dental caries, the most prevalent infectious disease world-wide. Biofilm formation by the cariogenic bacterium S. mutans is crucial for the pathogenesis of dental caries. Preventing or successfully treating severe caries remains an elusive task. The conventional treatment for dental caries usually involves the mechanical removal of dental biofilms, the use of antibiotics, or invasive procedures. However, these treatments are sometimes ineffective and the use of antibiotics or invasive procedures can result in the development of drug resistant pathogens or complications. Therefore, an alternative and less invasive approach is needed. Interestingly, the oral cavity exhibits higher concentrations of nitrite than other body sites. Elevated concentrations of nitrite in the oral cavity have been associated with a reduced prevalence of dental caries, suggesting that nitrite may be an alternative therapeutic for the treatment of caries. However, the exact mechanism(s) of how nitrite interferes with the biofilm formation and pathogenesis of S. mutans is unknown. The oral cavity harbors hundreds of bacterial species and they interact with each other and contribute to health and disease status of the oral cavity. Our preliminary data demonstrate that the hydrogen peroxide-producing oral commensal Streptococcus parasanguinis, inhibits S. mutans growth and biofilm formation in the presence of nitrite, revealing a new anti-infection strategy by the commensal oral streptococcus. In order to develop nitrite-containing therapeutics to treat dental caries, it is critical to understand mechanisms of S. mutans nitrite resistance. Transposon mutagenesis of S. mutans identified mutants that are resistant to S. parasanguinis and nitrite-mediated activity. One mutant is mapped to a gene coding for a histidine kinase (SMU.486). This histidine kinase and its response regulator (SMU.487) are homologous to a nitrite sensing two-component regulatory system in Escherichia coli, suggesting that they may mediate inhibition of S. mutans by S. parasanguinis and nitrite. Therefore, the immediate goal of this research proposal is to investigate how the nitrite sensing two-component system SMU.486 and SMU.487 mediate S. mutans? response to nitrosative stress and identify nitrosative stress response pathways controlled by SMU.486 and SMU.487. RNA-sequencing/transcriptomics will be used to analyze how SMU.486 and SMU.487 contribute to nitrosative stress resistance. In addition, the proposal will examine the ability of S. parasanguinis and nitrite-mediated activity to inhibit S. mutans and prevent caries in an animal model of dental caries. During this time the candidate will complete mentored training in Oral Microbiology, RNA sequencing/transcriptomics, and other professional development activities. The mentored training will prepare the candidate for the independent R00 phase in which the candidate will determine the suitability of S. parasanguinis as a probiotic in addition to continuing the characterization of other transposon mutants that confer resistance to S. parasanguinis and nitrite-mediated activity. The University of Alabama at Birmingham provides an exceptional environment for the candidate to receive mentored training and complete the outlined research due to the collaborative research environment, and state of the art facilities.